High Biomass Sorghum Hybrid ES5200

ABSTRACT

A hybrid sorghum, designated ES5200 is disclosed. The invention relates to the seeds of hybrid sorghum ES5200, to the plants of hybrid sorghum ES5200 and to methods for producing a sorghum plant by crossing the hybrid ES5200 with itself or another sorghum plant that is not a plant of sorghum hybrid ES5200. The invention further relates to hybrid sorghum seeds and plants. The invention further relates to methods for producing a sorghum plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other hybrid sorghum derived from sorghum hybrid ES5200.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application of and claims priorityto U.S. Provisional Patent Application No. 61/260,067, filed on Nov. 11,2009, which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive sorghum hybrid,designated ES5200. All publications cited in this application are hereinincorporated by reference.

The new sorghum hybrid ES5200 can be used as a biomass feedstock cropfor biofuel and/or biopower applications. Sorghum hybrid ES5200 may begrown for biomass, sugar, and/or forage.

As a biomass crop, sorghum can be of any of the many varieties whichhave a high biomass. Many of these varieties are photoperiod sensitiveand do not flower when grown in temperate latitudes. The continuousgrowth of vegetative biomass uninterrupted by flowering allows thesevarieties to produce significantly more vegetative biomass in comparisonto grain sorghums. These high biomass sorghum varieties and hybrids alsocomprise genetic backgrounds that have been selected for based onincreased plant height, increased biomass, increased growth rate, andlack of dwarfing genes, among other traits.

As a sugar crop, sorghum can be of any of the many varieties which havea high sugar content. Stalks are used for producing biofuel by squeezingthe juice and then fermenting the juice into a liquid biofuel, such asethanol. Varieties of sorghum grown for sugar content can beopen-pollinated varieties or hybrids. In addition to squeezing stalksfor juice, sorghum biomass may be treated with chemicals or industrialprocesses to release simple and complex sugars which can then be used inbiofuel production. Varieties of sorghum grown for sugar content can becharacterized by high brix values or by other more refined measurementsof sugar components.

As a forage crop, the genus Sorghum includes three principal distinctmorphotypes that are used as forages: forage sorghums, sudangrass, andsorghum×sudangrass hybrids. These three morphotypes have grosslydifferent phenotypes and different modes of principal utilization.Forage sorghums have very coarse stems and wide leaves, similar to corn(Zea mays L.), very low tillering capacity, and very slow speed ofregrowth after cutting. Consequently they are used nearly exclusively asa silage crop, never for hay production and only occasionally as directpasture. Sudangrass in comparison is very grassy, characterized by veryfine stems and narrow leaf blades, profuse tiller development, andexceptionally rapid recovery after cutting or grazing. Sudangrass(Sorghum bicolor ssp. sudanense L.) is an important forage species forpasture, grazing, green chop silage, hay and seed. Sudangrass is alsoreferred to by the scientific name sorghum×drummondii (Steudel) Millsp.& Chase (=S. bicolor×S. arundinaceum) (R. F. Barnes and J. B. Beard(ed.), Glossary of Crop Science Terms, Crop Science Society of America,July 1992, pg. 84). Classification and species relationships of sorghumand sudangrass are discussed in several reports (Harlan and deWet, 1972;Celarier, 1958). For a comprehensive review of the floralcharacteristics, plant culture, and methods of self-pollinating orhybridizing sudangrass, see Shertz and Dalton, Sorghum 41:577-588, InHybridization of Crop Plants, Fehr et al. (ed.), American Society ofAgronomy Inc. (1980). Sorghum×sudangrass hybrids (Sorghum bicolor×S.bicolor spp. sudanese) which result from crossing a sorghum female witha sudangrass male are generally intermediate in character expressionbetween sorghum and sudangrass. Sorghum×sudangrass hybrids are alsocommonly referred to as sorghum-sudangrass hybrids, sorghum/sudangrass,and SUDAX. Adding somewhat to the confusion of the nomenclature, thoseskilled in the art sometimes refer to sorghum×sudangrass hybrids as“sudangrass hybrids”. See, e.g., Miller and Stroup, 2003.

As a grain crop, Sorghum bicolor (L) Moench, is the fifth most importantcereal after rice, wheat, maize, and barley. It constitutes the mainfood grain for over 750 million people who live in the semi-arid tropicsof Africa, Asia, and Latin America. The largest group of producers aresmall-scale subsistence farmers with minimal access to production inputssuch as fertiliser(s), pesticides, improved seeds (hybrids orvarieties), good soil and water and improved credit facilities for theirpurchase.

There are many types of sorghum ranging in seed colour from whitethrough red to brown. Traditional types are open-pollinated from whichrural farmers retain seed for planting in the next season. Grain yieldstend to be lower than the modern hybrids which are slowly beingintroduced. Commercial production of hybrid seed is a problem in manydeveloping countries, and some rural farmers do not appreciate thatharvested hybrid grain cannot be retained for planting the next season.Therefore they find sorghum production from hybrid seed expensive, eventhough the grain yields are higher than the land races. Resource-poorfarmers prefer varieties incorporating the characteristics of resistanceto insects, disease, drought, birds, and with acceptable yields of bothgrain for human consumption and fodder for livestock feed.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, higher biomassyield, higher sugar yield, improved composition traits, improvedconversion traits, resistance to diseases and insects, better stems androots, tolerance to low temperatures, and better agronomiccharacteristics on grain quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection, ora combination of these methods.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.Inbred sorghum lines may be field tested in target area(s) for seedproduction for three or more years.

These processes, which lead to the final step of marketing anddistribution of a sorghum hybrid, usually take from 8 to 12 years fromthe time the first cross is made and may rely on the development ofimproved breeding lines as precursors. Therefore, development of newcultivars and hybrids is a time-consuming process that requires preciseforward planning, efficient use of resources, and a minimum of changesin direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of sorghum plant breeding is to develop new, unique andsuperior sorghum lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by self-pollination andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing, and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same sorghum traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new sorghum cultivars.

The development of new sorghum lines requires the development andselection of sorghum lines, the crossing of these lines and selection ofsuperior hybrid crosses. The hybrid seed is produced by manual crossesbetween selected male-fertile parents or by using male sterilitysystems. Additional data on parental lines, as well as the phenotype ofthe hybrid, influence the breeder's decision whether to continue withthe specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Sorghum inbred lines are typically developed by first crossing twoparent plants which may or may not be inbred. The parents may havetraits which the breeder desires to combine. Typically, a few plants arechosen from the resulting segregating F2 population, and these plantsare self pollinated for several generations in combination withselecting for increased uniformity and/or increased homozygosity. Theresulting new inbred lines can then be tested with other inbred lines todetermine combining ability and suitability as parents for hybrids.Utilizing male sterile parental lines, sorghum hybrids may be made thathave two (single hybrid) or three (double hybrid) parents.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep, et. al, 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current lines and hybrids. In additionto showing superior performance, there must be a demand for a new lineand hybrid that is compatible with industry standards or which creates anew market. The introduction of a new line or hybrid will incuradditional costs to the seed producer, the grower, processor andconsumer; for special advertising and marketing, altered seed andcommercial production practices, and new product utilization. Thetesting preceding release of a new line or hybrid should take intoconsideration research and development costs as well as technicalsuperiority of the final cultivar.

Sorghum, Sorghum bicolor (L.) Moench, is an important and valuable crop.Thus, a continuing goal of plant breeders is to develop stable, highyielding sorghum lines and hybrids that are agronomically sound. Thereasons for this goal are obviously to maximize the amount of grain,biomass, sugar, biofuel/acre, and/or biopower/acre produced on the landused and to supply food and fuel for both animals and humans. Toaccomplish this goal, the sorghum breeder must select and developsorghum plants that have the traits that result in superior lines andhybrids.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel sorghum hybriddesignated ES5200. This invention thus relates to the seeds of sorghumhybrid ES5200, to the plants of sorghum hybrid ES5200, and to methodsfor producing a sorghum plant produced by crossing sorghum hybrid ES5200with itself or another sorghum plant.

Thus, any such methods using sorghum hybrid ES5200 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using sorghum hybridES5200 as a parent are within the scope of this invention.Advantageously, the sorghum hybrid could be used in crosses with other,different, sorghum plants to produce first generation (F₁) sorghumhybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides for single geneconverted plants of sorghum hybrid ES5200. The single transferred genemay preferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as herbicide resistance, insectresistance, resistance for bacterial, fungal, or viral disease, malefertility, male sterility, enhanced nutritional quality, and industrialusage. The single gene may be a naturally occurring sorghum gene or atransgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of sorghum hybrid ES5200. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing sorghum plant, and ofregenerating plants having substantially the same genotype as theforegoing sorghum plant. Preferably, the regenerable cells in suchtissue cultures will be embryo, protoplast, meristematic cell, callus,pollen, glume, panicle leaf, pollen, ovule, cotyledon, hypocotyl, root,root tip, pistil, anther, floret, seed, stalk and rachis. Still further,the present invention provides sorghum plants regenerated from thetissue cultures of the invention.

Grain sorghum is an important and valuable food and feed grain crop.Thus, a continuing goal of plant breeders is to develop stable highyielding sorghum hybrids that are agronomically sound. The reasons forthis goal are obvious to maximize the amount of grain produced on theland used and to supply food for both animals and humans.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Biomass. Biomass includes harvestable plant tissues such as leaves,stems, and reproductive structures, or all plant tissues such as leaves,stems, roots, and reproductive structures.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

Coding region/sequence. The region within a DNA molecule (i.e., betweenthe start and stop codons) that encodes the amino acid sequence of aprotein.

Disease resistance. As used herein, the term “disease resistance” isdefined as the ability of plants to restrict the activities of aspecified pest, such as an insect, fungus, virus, or bacterium.

Disease tolerance. As used herein, the term “disease tolerance” isdefined as the ability of plants to endure a specified pest (such as aninsect, fungus, virus, or bacterium) or an adverse environmentalcondition and still perform and produce in spite of this disorder.

Embryo. The embryo is the small plant contained within a mature seed.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the cultivar, except for the characteristics derivedfrom a converted gene.

Gene Converted (Conversion). Gene converted (conversion) plant refers toplants which are developed by backcrossing, genetic engineering ormutation wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more traits transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation.

Genotype. Refers to the genetic constitution of a cell or organism.

Germination. The emergence and development from the seed embryo of thoseessential structures of a seedling. Germination is indicative of theability to produce a normal plant under favorable conditions.

Herbicide resistance/tolerance. As used herein, herbicide resistancealso includes herbicide tolerance. Herbicide resistance/tolerance is theability of a plant to survive and reproduce following exposure to a doseof herbicide that would be lethal to a susceptible plant.

Increased biomass. As used herein, increased biomass refers to increasedplant height, increased fresh matter yield, increased dry matter yield,and/or increased tiller number.

Pest resistance. As used herein, the term “pest resistance” is definedas the ability of plants to restrict the activities of a specified pest,such as an insect or nematode.

Pest tolerance. As used herein, the term “pest tolerance” is defined asthe ability of plants to endure a specified pest (such as an insect or anematode) still perform and produce in spite of this disorder.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed oranthers have been removed. A seed or embryo that will produce the plantis also considered to be the plant.

Plant characteristic. A plant characteristic can be a morphological,physiological, agronomic, or genetic feature of a plant.

Plant growth. This term refers to the process by which plants increasein size and mass. The increase in the number and size of plant organs isdirectly associated with an increase in cell numbers and cell size,which involves cell division, growth, expansion and differentiation.Plants utilize sunlight, water, carbon dioxide and minerals inbiosynthesis to provide energy and substances required for growth. Plantgrowth can be generally divided into vegetative and reproductive growthin the life cycle.

Plant Height. Plant height in centimeters is taken from soil surface tothe tip of the extended panicle at harvest.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

DETAILED DESCRIPTION OF THE INVENTION

Hybrid sorghum ES5200 is a Sorghum bicolor (L.) Moench, originated froma cross conducted in Maricopa, Ariz. in 2008 by crossing two publicvarieties, the female parent ATx2752 with the male parent BTx623 toproduce an F₁ female parent. The F₁ female parent was then crossed withmale parent R07007 to produce this double cross hybrid. The double crosshybrid has stability in producing ES5200. Plants of sorghum hybridES5200 exhibited low variance with respect to leaf width at maturity andleaf sheath length at maturity. ES5200 seedlings show no variance withrespect to anthocyanin coloration on the coleoptile.

Sorghum hybrid ES5200 has the following morphologic and othercharacteristics (based primarily on data collected at Thousand Oaks,Calif.).

TABLE 1 VARIETY DESCRIPTION INFORMATION General Categories: Kind:Sorghum Male sterile cytoplasm: A-1 Use class: Biomass Plant:Coleoptile: Green Plant pigment: Red Stalk: Diameter: Mid-stout Numberof recessive height genes plant height genotype: 1 Tillers: FewSweetness: Insipid Degree of senescence: Nonsenescent Leaf (first leafbelow flag leaf): Width (relative to class): Moderate Color: Dark greenMargin: Wavy Attitude: Horizontal Ligule: Present Midrib color: White

This invention is also directed to methods for producing a sorghum plantby crossing a first parent sorghum plant with a second parent sorghumplant wherein either the first or second parent sorghum plant is thesorghum plant of sorghum hybrid ES5200. Further, both first and secondparent sorghum plants can come from sorghum hybrid ES5200. Therefore,any methods using sorghum hybrid ES5200 are part of this invention:selfing, backcrosses, hybrid breeding and crosses to populations. Anyplants produced using sorghum hybrid ES5200 as a parent are within thescope of this invention.

Still further, this invention also is directed to methods for producinga sorghum hybrid ES5200-derived sorghum plant by crossing sorghum hybridES5200 with a second sorghum plant and growing the progeny seed, andrepeating the crossing and growing steps with the sorghum hybridES5200-derived plant from 0 to 7 times. Thus, any such methods usingsorghum hybrid ES5200 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plants andplant parts produced using sorghum hybrid ES5200 as a parent are withinthe scope of this invention, including plants derived from sorghumhybrid ES5200.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which corn plants can be regenerated,embryo, protoplast, meristematic cell, callus, pollen, glume, panicleleaf, pollen, ovule, cotyledon, hypocotyl, root, root tip, pistil,anther, floret, seed, stalk and rachis and the like.

Further Embodiments of the Invention Transformation Techniques

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar.

Culture for expressing desired structural genes and cultured cells areknown in the art. Also as known in the art, sorghum is transformable andregenerable such that whole plants containing and expressing desiredgenes under regulatory control may be obtained. General descriptions ofplant expression vectors and reporter genes and transformation protocolscan be found in Gruber, et al., “Vectors for Plant Transformation,” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993)). Moreover GUSexpression vectors and GUS gene cassettes are available from Clone TechLaboratories, Inc. (Palo Alto, Calif.), while luciferase expressionvectors and luciferase gene cassettes are available from Pro Mega Corp.(Madison, Wis.). General methods of culturing plant tissues are providedfor example by Mild, et al., “Procedures for Introducing Foreign DNAinto Plants” in Methods in Plant Molecular Biology and Biotechnology,Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993));and by Phillips, et al., “Cell-Tissue Culture and In-Vitro Manipulation”in Corn & Corn Improvement, 3rd Edition, Sprague, et al., (Eds., pp.345-387, American Society of Agronomy Inc. (1988)). Methods ofintroducing expression vectors into plant tissue include the directinfection or co-cultivation of plant cells with Agrobacteriumtumefaciens, described for example by Horsch, et al., Science, 227:1229(1985). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably expression vectors are introduced intoplant tissues using a microprojectile media delivery system with abiolistic device or using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed sorghum plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the sorghum plant(s).

Expression Vectors for Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford, et al.,Plant Physiol., 86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86(1987); Svab, et al., Plant Mol. Biol., 14:197 (1990); Hille, et al.,Plant Mol. Biol., 7:171 (1986). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate, or bromoxynil.Comai, et al., Nature, 317:741-744 (1985); Gordon-Kamm, et al., PlantCell, 2:603-618 (1990); and Stalker, et al., Science, 242:419-423(1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase. Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. U.S.A.,84:131 (1987); DeBlock, et al., EMBO J., 3:1681 (1984). Another approachto the identification of relatively rare transformation events has beenuse of a gene that encodes a dominant constitutive regulator of the Zeamays anthocyanin pigmentation pathway. Ludwig, et al., Science, 247:449(1990).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes Publication2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J. Cell Biol.,115:151 a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”preferred.” Promoters which initiate transcription only in certaintissue are referred to as “tissue-specific.” A “cell type” specificpromoter primarily drives expression in certain cell types in one ormore organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

The choice of promoter regions to be included in a recombinant constructdepends upon several factors, including, but not limited to, efficiency,selectability, inducibility, desired expression level, and cell- ortissue-preferential expression. It is a routine matter for one of skillin the art to modulate the expression of a coding sequence byappropriately selecting and positioning promoter regions relative to thecoding sequence. Transcription of a nucleic acid can be modulated in asimilar manner.

Some suitable promoter regions initiate transcription only, orpredominantly, in certain cell types. Methods for identifying andcharacterizing regulatory regions in plant genomic DNA are known,including, for example, those described in the following references:Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell,1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier etal., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology,110:1069-1079 (1996).

Examples of various classes of promoter regions are described below.Some of the promoter regions indicated below as well as additionalpromoter regions are described in more detail in U.S. Patent ApplicationSer. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691;60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569; 11/058,689;11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891; 11/097,589;11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;PCT/US05/011105; PCT/U505/23639; PCT/US05/034308; PCT/US05/034343; andPCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.

For example, the sequences of promoter regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837,YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121,YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110,YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156,PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377,PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, PT0743and YP0096 are set forth in the sequence listing of PCT/US06/040572; thesequence of promoter region PT0625 is set forth in the sequence listingof PCT/US05/034343; the sequences of promoter regions PT0623, YP0388,YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in the sequencelisting of U.S. patent application Ser. No. 11/172,703; the sequence ofpromoter region PR0924 is set forth in the sequence listing ofPCT/US07/62762; and the sequences of promoter regions p530c10,pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in the sequencelisting of PCT/US06/038236.

It will be appreciated that a promoter region may meet criteria for oneclassification based on its activity in one plant species, and yet meetcriteria for a different classification based on its activity in anotherplant species.

Broadly Expressing Promoters

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues. Forexample, a broadly expressing promoter can promote transcription of anoperably linked sequence in one or more of the shoot, shoot tip (apex),and leaves, but weakly or not at all in tissues such as roots or stems.As another example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of the stem,shoot, shoot tip (apex), and leaves, but can promote transcriptionweakly or not at all in tissues such as reproductive tissues of flowersand developing seeds. Non-limiting examples of broadly expressingpromoters that can be included in the nucleic acid constructs providedherein include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

Root Promoters

Root-active promoters confer transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., confer transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660,PT0683, and PT0758 promoters. Other root-preferential promoters includethe PT0613, PT0672, PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

Maturing Endosperm Promoters

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin promoter (Bustos et al., Plant Cell,1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturasepromoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), thesoybean a′ subunit of f3-conglycinin promoter (Chen et al., Proc. Natl.Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al.,Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kDzein promoter and 27 kD zein promoter. Also suitable are the Osgt-1promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell. Biol.,13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordeinpromoter. Other maturing endosperm promoters include the YP0092, PT0676,and PT0708 promoters.

Ovary Tissue Promoters

Promoters that are active in ovary tissues such as the ovule wall andmesocarp can also be useful, e.g., a polygalacturonidase promoter, thebanana TRX promoter, the melon actin promoter, YP0396, and PT0623.Examples of promoters that are active primarily in ovules includeYP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,YP0119, YP0120, and YP0374.

Embryo Sac/Early Endosperm Promoters

To achieve expression in embryo sac/early endosperm, regulatory regionscan be used that are active in polar nuclei and/or the central cell, orin precursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

Embryo Promoters

Regulatory regions that preferentially drive transcription in zygoticcells following fertilization can provide embryo-preferentialexpression. Most suitable are promoters that preferentially drivetranscription in early stage embryos prior to the heart stage, butexpression in late stage and maturing embryos is also suitable.Embryo-preferential promoters include the barley lipid transfer protein(Ltpl) promoter (Plant Cell Rep (2001) 20:647-654), YP0097, YP0107,YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, andPT0740.

Photosynthetic Tissue Promoters

Promoters active in photosynthetic tissue confer transcription in greentissues such as leaves and stems. Most suitable are promoters that driveexpression only or predominantly in such tissues. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778(1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al.,Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luanet al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphatedikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad.Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcbl*2 promoter (Cerdan etal., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570(1995)), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissuepromoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

Vascular Tissue Promoters

Examples of promoters that have high or preferential activity invascular bundles include YP0087, YP0093, YP0108, YP0022, and YP0080.Other vascular tissue-preferential promoters include the glycine-richcell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell,3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV)promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and therice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.Acad. Sci. USA, 101(2):687-692 (2004)).

Inducible Promoters

Inducible promoters confer transcription in response to external stimulisuch as chemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.Examples of drought-inducible promoters include YP0380, PT0848, YP0381,YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384,PT0688, YP0286, YP0377, PD1367, and PD0901. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PRO924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

Stem Promoters

A stem promoter may be specific to one or more stem tissues or specificto stem and other plant parts. Stem promoters may have high orpreferential activity in, for example, epidermis and cortex, vascularcambium, procambium, or xylem. Examples of stem promoters include YP0018which is disclosed in US20060015970 and CryIA(b) and CryIA(c) (Braga etal. 2003, Journal of New Seeds 5:209-221).

Other Promoters

Other classes of promoters include, but are not limited to,shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. Promotersdesignated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, andYP0096, as described in the above-referenced patent applications, mayalso be useful.

Other Regulatory Regions

A 5′ untranslated region (UTR) can be included in nucleic acidconstructs described herein. A 5′ UTR is transcribed, but is nottranslated, and lies between the start site of the transcript and thetranslation initiation codon and may include the +1 nucleotide. A 3′ UTRcan be positioned between the translation termination codon and the endof the transcript. UTRs can have particular functions such as increasingmRNA stability or attenuating translation. Examples of 3′ UTRs include,but are not limited to, polyadenylation signals and transcriptiontermination sequences, e.g., a nopaline synthase termination sequence.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129(1989); Fontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al.,Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al., J. Cell. Biol.,108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991); Kalderon, etal., Cell, 39:499-509 (1984); Steifel, et al., Plant Cell, 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is sorghum. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp.269-284 (1993)). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques.

Through the transformation of sorghum, the expression of genes can bealtered to enhance disease resistance, insect resistance, herbicideresistance, agronomic quality and other traits. Transformation can alsobe used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to sorghum as well as non-nativeDNA sequences can be transformed into sorghum and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well-known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as mu (Vicki Chandler, The Maize Handbook,ch. 118, Springer-Verlag (1994)) or other genetic elements such as aFRT, Lox, or other site specific integration site, antisense technology(see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809 (1988); and U.S. Pat.Nos. 5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor,Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech., 8(12):340-344(1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finnegan, et al.,Bio/Technology, 12: 883-888 (1994); and Neuhuber, et al., Mol. Gen.Genet., 244:230-241 (1994)); RNA interference (Napoli, et al., PlantCell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev.,13:139-141 (1999); Zamore, et al., Cell, 101:25-33 (2000); andMontgomery, et al., PNAS USA, 95:15502-15507 (1998)); virus-induced genesilencing (Burton, et al., Plant Cell, 12:691-705 (2000); and Baulcombe,Curr. Op. Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes(Haseloff, et al., Nature, 334: 585-591 (1988)); hairpin structures(Smith, et al., Nature, 407:319-320 (2000); WO 99/53050; and WO98/53083); MicroRNA (Aukerman & Sakai, Plant Cell, 15:2730-2741 (2003));ribozymes (Steinecke, et al., EMBO J., 11:1525 (1992); and Perriman, etal., Antisense Res. Dev., 3:253 (1993)); oligonucleotide mediatedtargeted modification (e.g., PCT Publication Nos. WO 03/076574 and WO99/25853); Zn-finger targeted molecules (e.g., PCT Publication Nos. WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant cultivar can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae); McDowell & Woffenden, TrendsBiotechnol., 21(4): 178-83 (2003); and Toyoda, et al., Transgenic Res.,11 (6):567-82 (2002).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser, et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

C. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Molec. Biol., 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See, PCT Appl. No. US93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of sorghum cysteine proteinaseinhibitor); Huub, et al., Plant Molec. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani, etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, PCTPublication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Molec. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Molec. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Molec. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See, PCT Publication No. WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT Publication No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci, 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor, et al., Abstract #497, Seventh International Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland (1994))(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/Technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant 1, 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/Technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

S. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2)(1995); Pieterse & Van Loon, Curr. Opin. Plant Bio., 7(4):456-64 (2004)and Somssich, Cell, 113(7):815-6 (2003).

T. Antifungal genes. See, Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs, et al., Planta, 183:258-264 (1991) andBushnell, et al., Can. J. of Plant Path., 20(2):137-149 (1998). Seealso, U.S. Pat. No. 6,875,907.

U. Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone, and their structurally relatedderivatives. For example, see U.S. Pat. No. 5,792,931.

V. Cystatin and cysteine proteinase inhibitors. See U.S. Pat. No.7,205,453.

W. Defensin genes. See PCT Publication No. WO 03/000863 and U.S. Pat.No. 6,911,577.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO 1, 7:1241 (1988) and Miki, et al., Theor. Appl. Genet., 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC Accession No. 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. EuropeanPat. Appl. No. 0 333 033 to Kumada, et al. and U.S. Pat. No. 4,975,374to Goodman, et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean Pat. Appl. No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/Technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for PAT activity.Exemplary of genes conferring resistance to phenoxy propionic acids andcyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2,and Acc1-S3 genes described by Marshall, et al., Theor. Appl. Genet.,83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) or a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, such as:

A. Modified lignin content, for example, by introducing or transforminga plant with genes that alter lignin content. See, Li, X., et al., PlantJ. (2008) 54(4): 569-581 and Saballos, A., et al., Genetics (2009)181(2): 783-795.

B. Modified plant height, for example, by introducing or transforming aplant with genes that alter plant height (Dw3 and dw3). See, Brown, P.J., et al., Genetics. (2008) 180(1) 629-637.

C. Modified profused wax and cuticular features, for example, byintroducing or transforming plants with genes that alter the cuticle/waxpathway for improvement of abiotic stress tolerance. See, Burrow, G. B.,et al., Theor. Appl. Genet. (2009) 118(3): 423-431 and McIntyyre, C. L.,et al., Genome (2008) 51(7): 524-533.

D. Modified diurnal oscillation, for example, by introducing ortransforming a plant with an antisense gene of starch branching enzyme(SBE). See, Mutisya, J., et al., J. Plant Physiol. (2009) 166(4):428-434.

E. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon, et al., Proc. Natl. Acad. Sci.USA., 89:2624 (1992).

F. Decreased phytate content. 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see, Van Hartingsveldt, et al., Gene,127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene; 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See, Raboy, et al., Maydica, 35:383 (1990).

G. Modified cell wall composition effected, for example, by transformingplants with a gene coding for an enzyme that alters the cell wallcomponents, such as but not limited to lignin, cellulose, xylan,hemicellulose, and saccharides. See, WO2008/069878 and US 2009-0070899A1, for examples of genes that can be used to alter cell wallcomposition.

4. Male Sterility:

Genes that Control Male Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describes a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on,”the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

A. A dominant nuclear gene, Ms(tc) controlling male sterility. See,Elkonin, L. A., Theor. Appl. Genet. (2005) 111(7): 1377-1384.

B. A tapetum-specific gene, RTS, a sorghum anther-specific gene isrequired for male fertility and its promoter sequence directstissue-specific gene expression in different plant species. Luo, Hong,et. al., Plant Molecular Biology., 62(3): 397-408(12) (2006).Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See International Publication No. WO 01/29237.

C. Introduction of various stamen-specific promoters. Anther-specificpromoters which are of particular utility in the production oftransgenic male-sterile monocots and plants for restoring theirfertility. See, U.S. Pat. No. 5,639,948. See also, InternationalPublication Nos. WO 92/13956 and WO 92/13957.

D. Introduction of the barnase and the barstar genes. See, Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

Alteration of Male Sterility Systems

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341, 6,297,426, 5,478,369,5,824,524, 5,850,014, and 6,265,640. See also, Hanson, Maureen R., et.al., “Interactions of Mitochondrial and Nuclear Genes That Affect MaleGametophyte Development,” Plant Cell., 16:S154-S169 (2004), all of whichare hereby incorporated by reference.

A. Modification of RNA editing within mitochondrial open reading frames.See, Pring, D. R., et al, Curr. Genet. (1998) 33(6): 429-436; Pring, D.R., et al., J. Hered. (1999) 90(3): 386-393; Pring, D. R., et al., Curr.Genet. (2001) 39(5-6): 371-376; and Hedgcoth, C., et al., Curr. Genet.(2002) 41(5): 357-365.

B. Cytoplasmic male sterility (CMS) from mutations at atp6 codons. See,Kempken, F., FEBS. Lett. (1998): 441(2): 159-160.

C. Inducing male sterility through heat shock. See, Wang, L., Yi ChuanXue Bao. (2000) 27(9): 834-838.

D. Inducing male sterility through treatment of streptomycin on sorghumcallus cultures. See, Elkonin, L. A., et al., Genetica (2008) 44(5):663-673.

5. Genes that Create a Site for Site Specific DNA Integration:

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., Site-Specific Recombination forGenetic Engineering in Plants, Plant Cell Rep, 21:925-932 (2003) andInternational Publication No. WO 99/25821, which are hereby incorporatedby reference. Other systems that may be used include the Gin recombinaseof phage Mu (Maeser, et al. (1991); Vicki Chandler, The Maize Handbook,ch. 118, Springer-Verlag (1994), the Pin recombinase of E. coli(Enomoto, et al. (1983)), and the R/RS system of the pSR1 plasmid(Araki, et al. (1992)).

6. Genes that Affect Abiotic Stress Resistance:

Genes that affect abiotic stress resistance (including, but not limitedto, flowering, panicle/glume and seed development, enhancement ofnitrogen utilization efficiency, altered nitrogen responsiveness,drought resistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress.

A. Modified drought stress tolerance, for example, by introducing ortransforming a plant with genes conferring drought stress tolerance.See, Srinivas, G., et al., Theor. Appl. Genet. (2009) February; 118(4):703-717.

B. Modified salt tolerance, for example, by introducing or transformingplants with genes that confer tolerance to salt. See, Li, Y., Pak. J.Biol. Sci. (2008) May; 11(9): 1268-1272.

C. Modified cold tolerance, for example by introducing or transformingplants with genes that confer cold tolerance. See, Knoll, J., et al.,Theor. Appl. Genet. (2008) 116(4): 577-587 and Knoll, J., et al., Theor.Appl. Genet. (2008) 116(4): 541-543.

For example, see: International Publication No. WO 00/73475 where wateruse efficiency is altered through alteration of malate; U.S. Pat. Nos.5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446,6,717,034, and 6,801,104, and International Publication Nos. WO2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO2004/076638, WO 98/09521, and WO 99/38977 describing genes, includingCBF genes and transcription factors effective in mitigating the negativeeffects of freezing, high salinity, and drought on plants, as well asconferring other positive effects on plant phenotype; U.S. PublicationNo. 2004/0148654 and International Publication No. WO 01/36596 whereabscisic acid is altered in plants resulting in improved plant phenotypesuch as increased yield and/or increased tolerance to abiotic stress;International Publication Nos. WO 2000/006341 and WO 04/090143, U.S.Publication No. 2004/0237147, and U.S. Pat. No. 6,992,237, wherecytokinin expression is modified resulting in plants with increasedstress tolerance, such as drought tolerance and/or increased yield. Alsosee, International Publication Nos. WO 02/02776, WO 2003/052063, WO01/64898, JP 2002281975, and U.S. Pat. Nos. 6,084,153, 6,177,275, and6,107,547 (enhancement of nitrogen utilization and altered nitrogenresponsiveness). For ethylene alteration, see, U.S. Publication Nos.2004/0128719 and 2003/0166197 and International Publication No. WO2000/32761. For plant transcription factors or transcriptionalregulators of abiotic stress, see, e.g., U.S. Publication Nos.2004/0098764 and 2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth, and/or plantstructure, can be introduced or introgressed into plants, see, e.g.,International Publication Nos. WO 97/49811 (LHY), WO 98/56918 (ESD4), WO97/10339, WO 96/14414 (CON), WO 96/38560, WO 01/21822 (VRN1), WO00/44918 (VRN2), WO 99/49064 (GI), WO 00/46358 (FR1), WO 97/29123, WO99/09174 (D8 and Rht), and U.S. Pat. Nos. 6,573,430 (TFL), 6,713,663(FT), 6,794,560, 6,307,126 (GAI), and International Publication Nos. WO2004/076638 and WO 2004/031349 (transcription factors).

Examples of exogenous nucleic acid sequences that can be used in themethods described herein include, but are not limited to, sequencesencoding genes or fragments thereof that modulate cold tolerance, frosttolerance, heat tolerance, drought tolerance, water used efficiency,nitrogen use efficiency, pest resistance, herbicide resistance, biomass,chemical composition, plant architecture, biopower conversionproperties, and/or biofuel conversion properties. In particular,exemplary sequences are described in the following applications whichare incorporated herein by reference in their entirety: US20080131581,US20080072340, US20070277269, US20070214517, US 20070192907, US20070174936, US 20070101460, US 20070094750, US20070083953, US20070061914, US20070039067, US20070006346, US20070006345, US20060294622,US20060195943, US20060168696, US20060150285, US20060143729,US20060134786, US20060112454, US20060057724, US20060010518,US20050229270, US20050223434, and US20030217388.

Methods for Sorghum Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Mild, et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation,” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch, et al., Science,227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci., 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber, et al., supra, Miki, et al., supra;Moloney, et al., Plant Cell Reports, 8:238 (1989);Agrobacterium-mediated transformation of sorghum is provided by Zhao, Z.Y., et al., Plant Mol. Biol. (2000) 44(6): 789-798 and Gurel, et al.,Plant Cell Rep. (2009) 28(3): 429-4244. See also, U.S. Pat. No.5,591,616, issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some major cereal cropspecies and gymnosperms have generally been recalcitrant to this mode ofgene transfer, even though some success has recently been achieved insorghum and corn. Hiei, et al., The Plant Journal, 6:271-282 (1994) andU.S. Pat. No. 5,591,616, issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. See, Casas, M.,et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90(23): 11212-11216, fortransformation of sorghum plants via microprojectile bombardment;Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/Technology, 6:559-563(1988); Sanford, J. C., Physiol Plant, 7:206 (1990); Klein, et al.,Biotechnology, 10:268 (1992). In corn, several target tissues can bebombarded with DNA-coated microprojectiles in order to producetransgenic plants, including, for example, callus (Type I or Type II),immature embryos, and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology, 9:996 (1991). Additionally,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes, et al., EMBO 1, 4:2731 (1985); Christou,et al., Proc Natl. Acad. Sci. U.S.A., 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn, et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin, et al., Plant Cell, 4:1495-1505 (1992); and Spencer, et al.,Plant Mol. Biol., 24:51-61 (1994).

Following transformation of sorghum target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues, and/or plants usingregeneration and selection methods now well known in the art.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such as IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARS), AmplifiedFragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs),which are also referred to as Microsatellites, and Single NucleotidePolymorphisms (SNPs). For example, see, Yonemaru, J., et al.,“Development of genome-wide simple sequence repeat markers usingwhole-genome shotgun sequences of sorghum (Sorghum bicolor (L.) Moench)”DNA Res. (2009) 16(3): 187-193; Duan Y., et al., “Construction ofmethylation linkage map based on MSAP and SSR markers in Sorghum bicolor(L.). IUMBMB Life (2009) 61(6): 663-669; Srinivas, G. et al.,“Identification of quantitative trait loci for agronomically importanttraits and their association with genic-microsatellite markers insorghum” Theor. App. Genet. (2009) 118(8): 1439-1454; Mace, E. S., etal., “A consensus genetic map of sorghum that integrates multiplecomponent maps and high-throughput Diversity Array Technology (DArT)markers” BMC Plant Biol. (2009) 9:13; Cregan et. al, “An IntegratedGenetic Linkage Map of the Soybean Genome,” Crop Science, 39:1464-1490(1999), and Berry, et al., “Assessing Probability of Ancestry UsingSimple Sequence Repeat Profiles: Applications to Maize Inbred Lines andSoybean Varieties,” Genetics, 165:331-342 (2003), each of which areincorporated by reference herein in their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forsorghum hybrid ES5200.

Primers and PCR protocols for assaying these and other markers arewidely known in the art. In addition to being used for identification ofsorghum hybrid ES5200 and plant parts and plant cells of sorghum hybridES5200, the genetic profile may be used to identify a sorghum plantproduced through the use of sorghum hybrid ES5200 or to verify apedigree for progeny plants produced through the use of sorghum hybridES5200. The genetic marker profile is also useful in breeding anddeveloping backcross conversions.

The present invention comprises a sorghum plant characterized bymolecular and physiological data obtained from the representative sampleof said inbred line and or hybrid deposited with the American TypeCulture Collection (ATCC). Further provided by the invention is asorghum hybrid plant formed by the combination of the disclosed sorghumhybrid plant or plant cell with another sorghum plant or cell.

Means of performing genetic marker profiles using SSR polymorphisms arewell-known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing hybrids or varieties it is preferable ifall SSR profiles are performed in the same lab.

Primers used may be publicly available and may be found in for examplein U.S. Pat. Nos. 7,232,940, 7,217,003, 7,250,556, 7,214,851, 7,195,887,and 7,192,774.

In addition, plants and plant parts substantially benefiting from theuse of sorghum line ES5200 in their development, such as sorghum lineES5200 comprising a backcross conversion, transgene, or geneticsterility factor, may be identified by having a molecular marker profilewith a high percent identity to sorghum hybrid ES5200. Such a percentidentity might be 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical tosorghum hybrid ES5200.

The SSR profile of sorghum hybrid ES5200 also can be used to identifyplants developed from the use of sorghum hybrid ES5200, as well as cellsand other plant parts thereof. Such plants may be developed using themarkers identified in International Publication No. WO 00/31964, U.S.Pat. No. 6,162,967 and U.S. application Ser. No. 09/954,773. Progenyplants and plant parts produced using sorghum hybrid ES5200 may beidentified by having a molecular marker profile of at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 99.5% genetic contribution from a sorghumhybrid or line, as measured by either percent identity or percentsimilarity. Unique molecular profiles may be identified with othermolecular tools such as SNPs and RFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such sorghum plant. Such unique SSR profilesmay arise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, a hybrid and progeny produced from such sorghum plant.

Gene Conversion

When the term “sorghum plant” is used in the context of the presentinvention, this also includes any gene conversions of that line. Theterm gene converted plant as used herein refers to those sorghum plantswhich are developed by a plant breeding technique called backcrossingwherein essentially all of the desired morphological and physiologicalcharacteristics of a cultivar are recovered in addition to the one ormore genes transferred into the cultivar via the backcrossing technique.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the line. The term backcrossing asused herein refers to the repeated crossing of a hybrid progeny back toone of the parental sorghum plants, the recurrent parent, for thatcultivar, i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times tothe recurrent parent. The parental sorghum plant which contributes thegene for the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental sorghum plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper (1994); Fehr (1987)). In a typical backcross protocol, theoriginal cultivar of interest (recurrent parent) is crossed to a secondcultivar (nonrecurrent parent) that carries the single gene or genes ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a sorghum plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to one or moretransferred genes from the nonrecurrent parent as determined at the 5%significance level when grown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more traits or characteristics in theoriginal cultivar. To accomplish this, one or more genes of therecurrent cultivar is modified or substituted with the desired gene orgenes from the nonrecurrent parent, while retaining essentially all ofthe rest of the desired genetic, and therefore the desired physiologicaland morphological, constitution of the original cultivar. The choice ofthe particular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic(s) beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic. Examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196, 5,948,957, and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Introduction of a New Trait or Locus into Sorghum Hybrid ES5200

Sorghum hybrid ES5200 represents a new base into which a new locus ortrait may be introgressed. Direct transformation and backcrossingrepresent two important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of Sorghum Hybrid ES5200

A backcross conversion of sorghum hybrid ES5200 occurs when DNAsequences are introduced through backcrossing (Hallauer, et al., “CornBreeding,” Corn and Corn Improvements, No. 18, pp. 463-481 (1988)), withsorghum hybrid ES5200 utilized as the recurrent parent. Both naturallyoccurring and transgenic DNA sequences may be introduced throughbackcrossing techniques. A backcross conversion may produce a plant witha trait or locus conversion in at least two or more backcrosses,including at least 2 crosses, at least 3 crosses, at least 4 crosses, atleast 5 crosses, and the like. Molecular marker assisted breeding orselection may be utilized to reduce the number of backcrosses necessaryto achieve the backcross conversion. For example, see, Openshaw, S. J.,et al., Marker-assisted Selection in Backcross Breeding, in: ProceedingsSymposium of the Analysis of Molecular Data, Crop Science Society ofAmerica, Corvallis, Oreg. (August 1994), where it is demonstrated that abackcross conversion can be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (See,Hallauer, et al., in Corn and Corn Improvement, Sprague and Dudley,Third Ed. (1998)). Desired traits that may be transferred throughbackcross conversion include, but are not limited to, sterility (nuclearand cytoplasmic), fertility restoration, nutritional enhancements,drought tolerance, nitrogen utilization, altered fatty acid profile, lowphytate, industrial enhancements, disease resistance (bacterial, fungalor viral), insect resistance, and herbicide resistance. In addition, anintrogression site itself, such as an FRT site, Lox site, or other sitespecific integration site, may be inserted by backcrossing and utilizedfor direct insertion of one or more genes of interest into a specificplant variety. In some embodiments of the invention, the number of locithat may be backcrossed into sorghum line ES5200 is at least 1, 2, 3, 4,or 5, and/or no more than 6, 5, 4, 3, or 2. A single locus may containseveral transgenes, such as a transgene for disease resistance that, inthe same expression vector, also contains a transgene for herbicideresistance. The gene for herbicide resistance may be used as aselectable marker and/or as a phenotypic trait. A single locusconversion of a site specific integration system allows for theintegration of multiple genes at the converted loci.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of sorghum andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T., et al., Crop Sci.,31:333-337 (1991); Stephens, P. A., et al., Theor. Appl. Genet.,82:633-635 (1991); Komatsuda, T., et al., Plant Cell, Tissue and OrganCulture, 28:103-113 (1992); Dhir, S., et al., Plant Cell Reports,11:285-289 (1992); Pandey, P., et al., Japan J. Breed., 42:1-5 (1992);and Shetty, K., et al., Plant Science, 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944, issued Jun. 18, 1991 to Collins, et al., andU.S. Pat. No. 5,008,200, issued Apr. 16, 1991 to Ranch, et al. Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce sorghum plants having the physiological andmorphological characteristics of sorghum hybrid ES5200.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are embryo, protoplast, meristematic cell,callus, pollen, glume, panicle leaf, pollen, ovule, cotyledon,hypocotyl, root, root tip, pistil, anther, floret, seed, stalk andrachis, and the like. Means for preparing and maintaining plant tissueculture are well known in the art. By way of example, a tissue culturecomprising organs has been used to produce regenerated plants. U.S. Pat.Nos. 5,959,185; 5,973,234, and 5,977,445 describe certain techniques,the disclosures of which are incorporated herein by reference.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which sorghum plants canbe regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as embryo, protoplast,meristematic cell, callus, pollen, glume, panicle leaf, pollen, ovule,cotyledon, hypocotyl, root, root tip, pistil, anther, floret, seed,stalk and rachis, and the like. Thus, another aspect of this inventionis to provide for cells which upon growth and differentiation produce acultivar having essentially all of the physiological and morphologicalcharacteristics of ES5200.

The present invention contemplates a sorghum plant regenerated from atissue culture of a plant of the present invention. As is well-known inthe art, tissue culture of sorghum can be used for the in vitroregeneration of a sorghum plant. Tissue culture of various tissues ofsorghum and regeneration of plants therefrom is well-known and widelypublished. For example, reference may be had to Chu, Q. R., et al., “Anovel basal medium for embryogenic callus induction of Southern UScrosses,” Rice Biotechnology Quarterly, 32:19-20 (1997); and Oono, K.,“Broadening the Genetic Variability By Tissue Culture Methods,” Jap. J.Breed., 33 (Suppl. 2), 306-307, illus. 1983. Thus, another aspect ofthis invention is to provide cells which upon growth and differentiationproduce sorghum plants having the physiological and morphologicalcharacteristics of hybrid ES5200.

Duncan, et al., Planta, 165:322-332 (1985), reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both cultivars and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports, 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two cultivars.Other published reports also indicated that “non-traditional” tissuesare capable of producing somatic embryogenesis and plant regeneration.K. P. Rao, et al., Maize Genetics Cooperation Newsletter, 60:64-65(1986), refers to somatic embryogenesis from glume callus cultures andB. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987), indicatessomatic embryogenesis from the tissue cultures of corn leaf segments.Thus, it is clear from the literature that the state of the art is suchthat these methods of obtaining plants are routinely used and have avery high rate of success.

Tissue culture of corn, for example, is described in European PatentApplication Publication 160,390. Corn tissue culture procedures are alsodescribed in Green and Rhodes, “Plant Regeneration in Tissue Culture ofMaize,” Maize for Biological Research, Plant Molecular BiologyAssociation, Charlottesville, Va., 367-372 (1982), and in Duncan, etal., “The Production of Callus Capable of Plant Regeneration fromImmature Embryos of Numerous Zea Mays Genotypes,” 165 Planta, 322:332(1985). Thus, another aspect of this invention is to provide cells whichupon growth and differentiation produce corn plants having thephysiological and morphological characteristics of sorghum hybridES5200.

Crossing Methods and Hybrid Production

One or more of the methods of breeding described herein can be used withthe sorghum hybrid described herein. In some embodiments, the sorghumhybrid described herein can be used as a parent to produce a differentsorghum plant. In some embodiments, the sorghum hybrid produced is ahigh biomass sorghum. In some embodiments, the sorghum hybrid describedherein can be used in breeding to develop a different sorghum plantthrough further selection exclusively among plants of the hybrid or bycrossing the hybrid with another sorghum plant and making furtherselections through selfed or backcrossed generations. Techniques such ashaploid doubling and marker assisted selection for homozygosity can beused to accelerate the process of inbreeding.

Sorghum plants are bred in most cases by self pollination techniques.With the incorporation of male sterility (either genetic or cytoplasmic)cross pollination breeding techniques can also be utilized. Sorghum hasa perfect flower with both male and female parts in the same flowerlocated in the panicle. The flowers are usually in pairs on the paniclebranches. Natural pollination occurs in sorghum when anthers (maleflowers) open and pollen falls onto receptive stigma (female flowers).Because of the close proximity of male (anthers) and female (stigma) inthe panicle, self pollination can be high. Cross pollination may occurwhen wind or convection currents move pollen from the anthers of oneplant to receptive stigma on another plant. Cross pollination is greatlyenhanced with incorporation of male sterility which renders male flowersnonviable without affecting the female flowers. Successful pollinationin the case of male sterile flowers requires cross pollination.

The development of sorghum hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding methods, and to a lesser extentpopulation breeding methods, are used to develop inbred lines frombreeding populations. Breeding programs combine desirable traits fromtwo or more inbred lines into breeding pools from which new inbred linesare developed by selfing and selection of desired phenotypes. The newinbreds are crossed with other inbred lines and the hybrids from thesecrosses are evaluated to determine which have commercial potential.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complement the other. If the two original parents donot provide all of the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically, in the pedigree method of breeding five or more generationsof selfing and selection is practiced. F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄to F₅, etc.

Backcrossing can be used to improve an inbred line. Backcrossingtransfers a specific desirable trait from one inbred or source to aninbred that lacks that trait. This can be accomplished for example byfirst crossing a superior inbred (A) (recurrent parent) to a donorinbred (non-recurrent parent), which carries the appropriate genes(s)for the trait in question. The progeny of this cross is then mated backto the superior recurrent parent (A) followed by selection in theresultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny will be heterozygous forloci controlling the characteristic being transferred, but will be likethe superior parent for most or almost all other genes. The lastbackcross generation would be selfed to give pure breeding progeny forthe gene(s) being transferred.

A hybrid sorghum variety can be the cross of two inbred lines, each ofwhich may have one or more desirable characteristics lacked by the otheror which complement the other. The hybrid progeny of the firstgeneration is designated F1. In the development of hybrids only the F1hybrid plants are sought. The hybrid is more vigorous than its inbredparents. This hybrid vigor, or heterosis, can be manifested in manyways, including increased vegetative growth and increased yield.

The development of a hybrid sorghum variety involves five steps: (1) theformation of “restorer” and “non-restorer” germplasm pools; (2) theselection of superior plants from various “restorer” and “non-restorer”germplasm pools; (3) the selfing of the superior plants for severalgenerations to produce a series of inbred lines, which althoughdifferent from each other, each breed true and are highly uniform; (4)the conversion of inbred lines classified as non-restorers tocytoplasmic male sterile (CMS) forms, and (5) crossing the selectedcytoplasmic male sterile (CMS) inbred lines with selected fertile inbredlines (restorer lines) to produce the hybrid progeny (F1).

Because sorghum is normally a self-pollinated plant and because bothmale and female flowers are in the same panicle, large numbers of hybridseed can only be produced by using cytoplasmic male sterile (CMS)inbreds. Inbred male sterile lines are developed by converting inbredlines to CMS. This is achieved by transferring the chromosomes of theline to be sterilized into sterile cytoplasm by a series of backcrosses,using a male sterile line as a female parent and the line to besterilized as the recurrent and pollen parent in all crosses. Afterconversion to male sterility the line is designated the (A) line. Lineswith fertility restoring genes cannot be converted into male sterileA-lines. The original line is designated the (B) line.

Flowers of the CMS inbred are fertilized with pollen from a male fertileinbred carrying genes which restore male fertility in the hybrid (F1)plants. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid between any two inbreds will alwaysbe the same. Once the inbreds that give the best hybrid have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained.

Where the hybrid plants produced are for use as forage orbiofuel/biopower feedstock, then it may not be necessary that theflowers of the CMS inbred are fertilized with pollen from a male fertileinbred carrying genes which restore male fertility in the hybrid (F1)plants. The flowers of the CMS inbred can fertilized with pollen from amale fertile inbred (such as a B line) chosen for combining ability withthe female parent inbred line. Since seed production may not benecessary or even desirable in a high biomass sorghum hybrid, there maybe no need to use a male inbred line that carries genes which restoremale fertility.

This invention also is directed to methods for producing a sorghum plantby crossing a first parent sorghum plant with a second parent sorghumplant wherein the first or second parent sorghum plant is a sorghumplant of ES5200. Further, both first and second parent sorghum plantscan come from the sorghum plant ES5200. Thus, any such methods using thesorghum hybrid ES5200 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using sorghum hybrid ES5200 as a parent are within the scope ofthis invention, including those derived from sorghum hybrid ES5200.Advantageously, the sorghum hybrid could be used in crosses with other,different, sorghum plants to produce the first generation (F₁) sorghumhybrid seeds and plants with superior characteristics. The line of theinvention can also be used for transformation where exogenous genes areintroduced and expressed by the line of the invention. Genetic variantscreated either through traditional breeding methods using ES5200 orthrough transformation of ES5200 by any of a number of protocols knownto those of skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with sorghumhybrid ES5200 in the development of further sorghum plants. One suchembodiment is a method for developing a ES5200 progeny sorghum plant ina sorghum plant breeding program comprising: obtaining the sorghumplant, or a part thereof, of hybrid ES5200 utilizing said plant or plantpart as a source of breeding material and selecting a ES5200 progenyplant with molecular markers in common with ES5200 and/or withmorphological and/or physiological characteristics selected from thecharacteristics listed in Tables 1, 2, 4 or 6. Breeding steps that maybe used in the sorghum plant breeding program include pedigree breeding,back crossing, mutation breeding, and recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example SSR markers)and the making of double haploids may be utilized.

Another method involves producing a population of hybrid ES5200 progenysorghum plants, comprising crossing hybrid ES5200 with another sorghumplant, thereby producing a population of sorghum plants, which, onaverage, derive 50% of their alleles from hybrid ES5200. A plant of thispopulation may be selected and repeatedly selfed or sibbed with asorghum cultivar resulting from these successive filial generations. Oneembodiment of this invention is the sorghum hybrid produced by thismethod and that has obtained at least 50% of its alleles from ES5200.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two different plant hybrids or varieties todetermine if there is no significant difference between the two traitsexpressed by the hybrids or varieties. For example, see, Fehr and Walt,Principles of Cultivar Development, pp. 261-286 (1987). Thus theinvention includes sorghum hybrid ES5200 progeny sorghum plantscomprising a combination of at least two ES5200 traits selected from thegroup consisting of those listed in Tables 1, 2, 4, and 6, or the ES5200combination of traits listed in the Summary of the Invention, so thatsaid progeny sorghum plant is not significantly different for saidtraits than sorghum hybrid ES5200 as determined at the 5% significancelevel when grown in the same environment. Using techniques describedherein, molecular markers may be used to identify said progeny plant asa ES5200 progeny plant. Mean trait values may be used to determinewhether trait differences are significant, and preferably the traits aremeasured on plants grown under the same environmental conditions. Oncesuch a line or hybrid is developed its value is substantial since it isimportant to advance the germplasm base as a whole in order to maintainor improve traits such as yield, disease resistance, pest resistance,and plant performance in extreme environmental conditions.

Progeny of sorghum hybrid ES5200 may also be characterized through theirfilial relationship with sorghum hybrid ES5200, as for example, beingwithin a certain number of breeding crosses of sorghum hybrid ES5200. Abreeding cross is a cross made to introduce new genetics into theprogeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween sorghum hybrid ES5200 and its progeny and any plants derivedfrom ES5200. For example, progeny produced by the methods describedherein may be within 1, 2, 3, 4, or 5 breeding crosses of sorghum hybridES5200.

In some embodiments, plants of the inbred sorghum hybrid ES5200 can beused as female parents to produce hybrids or as female or male parentsto produce new inbred lines. In some embodiments, the sorghum hybridES5200 can be bred with another sorghum plant (Sorghum bicolor) thatalso contributes to a high biomass phenotype in the resulting hybridsorghum plant. In other embodiments, the sorghum hybrid ES5200 can beused as a parent in breeding with exotic germplasm to make new hybridsand/or inbred lines. Examples of exotic germplasm are wild, weedy, orcultivated sorghum species, such as but not limited to, Sorghum almum,Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor(such as bicolor, guinea, caudatum, kafir, and durra), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum miliaceum, Sorghum nigrum,Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghumpurpureosericeum, Sorghum stipoideum, Sorghum sudanensese, Sorghumtimorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum,Sorghum vulgare, or hybrids such as Sorghum×almum, orSorghum×drummondii. In some embodiments, sorghum hybrid ES5200 can beused as a parent in breeding with other genera, such as Saccharum.

Single Cross

A single cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny. Much of the hybrid vigor exhibited by F₁ hybridsis lost in the next generation (F₂). Consequently, seed from hybridvarieties is not typically used for planting stock.

Hybrid sorghum can be produced using wind to move the pollen.Alternating strips of the cytoplasmic male sterile inbred (female) andthe male fertile inbred (male) are planted in the same field. Wind movesthe pollen shed by the male inbred to receptive stigma on the female.Providing that there is sufficient isolation from sources of foreignsorghum pollen, the stigma of the male sterile inbred (female) will befertilized only with pollen from the male fertile inbred (male). Theresulting seed, born on the male sterile (female) plants is thereforehybrid and will form hybrid plants that have full fertility restored. Insome embodiments, if the hybrid sorghum is used as forage or for biomassproduction, then it may be unnecessary to restore fertility.

Double Cross

A double cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny, which is then crossed with a third inbred line.This technique can be used to produce forage and high biomass sorghumhybrids. Such hybrids typically exhibit greater variability than singlecross hybrids. This variability can be an advantage in adaptabilityacross environments.

Top Cross

A top cross is a cross between a selection, line, clone etc., and acommon pollen parent which may be a variety, inbred line, single cross,etc. The common pollen parent is called the top cross or tester parent.This type of test cross involves mating a series of individuals to acommon parent to produce half-sib or full-sib families for evaluation.The test can be used to determine the general combining ability of anindividual. Typically, those individuals that perform well in thetestcross evaluation are advanced to trials where they are evaluated incrosses with other selected individuals. In sorghum, a top cross iscommonly an inbred variety cross. In some embodiments, where the topcross is between inbred lines, and the resulting hybrids evaluatedexhibit desirable traits, there may be no need for further testing anddevelopment, for example, where the resulting hybrids have a highbiomass phenotype. In some embodiments, where the top cross is betweeninbred lines, and the resulting hybrids evaluated exhibit sterility,there may be no need for further testing and development.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such assorghum hybrid ES5200 and another sorghum plant having one or moredesirable characteristics that is lacking or which complements sorghumhybrid ES5200. If the two original parents do not provide all thedesired characteristics, other sources can be included in the breedingpopulation. In the pedigree method, superior plants are selfed andselected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, asorghum line may be crossed with another sorghum line to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC₁ or BC₂.Progeny are selfed and selected so that the newly developed variety hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the nonrecurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new sorghumvarieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of sorghum hybrid ES5200, comprising the steps ofcrossing a plant of sorghum hybrid ES5200 with a donor plant comprisinga desired trait, selecting an F₁ progeny plant comprising the desiredtrait, and backcrossing the selected F₁ progeny plant to a plant ofsorghum hybrid ES5200. This method may further comprise the step ofobtaining a molecular marker profile of sorghum hybrid ES5200 and usingthe molecular marker profile to select for a progeny plant with thedesired trait and the molecular marker profile of sorghum hybrid ES5200.In one embodiment the desired trait is a mutant gene or transgenepresent in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Sorghum ES5200 is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny and selfedprogeny. The selected progeny are cross pollinated with each other toform progeny for another population. This population is planted andagain superior plants are selected to cross pollinate with each other.Recurrent selection is a cyclical process and therefore can be repeatedas many times as desired. The objective of recurrent selection is toimprove the traits of a population. The improved population can then beused as a source of breeding material to obtain new varieties forcommercial or breeding use, including the production of a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self pollination, directed pollinationcould be used as part of the breeding program.

Mutation Breeding

Mutation breeding is another method of introducing new traits intosorghum hybrid ES5200. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in“Principles of Cultivar Development,” Fehr, Macmillan Publishing Company(1993). In addition, mutations created in other sorghum plants may beused to produce a backcross conversion of sorghum hybrid ES5200 thatcomprises such mutation.

Breeding with Molecular Markers

Molecular markers may be used in plant breeding methods utilizingsorghum line ES5200.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. See, for example, Dinka, S. J., et al., “Predictingthe size of the progeny mapping population required to positionallyclone a gene,” Genetics., 176(4):2035-54 (2007); Gonzalez, C., et al.,“Molecular and pathogenic characterization of new Xanthomonas oryzaestrains from West Africa,” Mol. Plant. Microbe Interact., 20(5):534-546(2007); Jin, H., et al., “Molecular and cytogenic characterization of anOryza officinalis-O. sativa chromosome 4 addition line and itsprogenies,” Plant Mol. Biol., 62(4-5):769-777 (2006); Pan, G., et al.,“Map-based cloning of a novel sorghum cytochrome P450 gene CYP81A6 thatconfers resistance to two different classes of herbicides,” Plant Mol.Biol., 61(6):933-943 (2006); Huang, W., et al., “RFLP analysis formitochondrial genome of CMS-sorghum,” Journal of Genetics and Genomics.,33(4):330-338 (2007); and I. K. Vasil (ed.), DNA-based markers inplants, Kluwer Academic Press Dordrecht, the Netherlands.

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. See forexample, Yonemaru, Jun-ichi, et al., “Development of Genome-wide SimpleSequence Repeat Markers Using Whole-genome Shotgun Sequences of Sorghum(Sorghum bicolor (L.) Moench) DNA Research. Published online April 2009and Menz, Monica A., et al. “Genetic Diversity of Public Inbreds ofSorghum Determined by Mapped AFLP and SSR Markers” Crop Science.44:1236-1244 (2004). Single Nucleotide Polymorphisms may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Sorghum DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. See, Paterson, A. H., Int. J.Plant Genomics (2008) 2008:362451; Rouline A., et al., “Whole genomesurveys of rice, maize and sorghum reveal multiple horizontal transfersof the LTR-retrotransposon Route66 in Poaceae” BMC Evol. Biol. (2009)9:58; Paterson, A. H., et al., “The Sorghum bicolor genome and thediversification of grasses” Nature (2009) 457(7229): 551-556; Sasaki,T., et al., “Plant genomics: sorghum in sequence” Nature (2009)457(7229): 547-548.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See Winn, J. A., et al. (2009) “QTL mapping of a high proteindigestibility trait in Sorghum bicolor” Int. J. Plant Genomics,2009:471853,. Epub. 2009 Jul. 7.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a sorghum plant for which sorghum line ES5200 is a parent canbe used to produce double haploid plants. Double haploids are producedby the doubling of a set of chromosomes (1N) from a heterozygous plantto produce a completely homozygous individual. For example, see, Wan, etal., “Efficient Production of Doubled Haploid Plants Through ColchicineTreatment of Anther-Derived Maize Callus,” Theoretical and AppliedGenetics, 77:889-892 (1989), and U.S. Pat. No. 7,135,615.

Methods for obtaining haploid plants are also disclosed in Kobayashi,M., et al., Journ. of Heredity, 71(1):9-14 (1980), Pollacsek, M.,12(3):247-251, Agronomie, Paris (1992); Cho-Un-Haing, et al., Journ. ofPlant Biol., 39(3):185-188 (1996); Verdoodt, L., et al., 96(2):294-300(February 1998); Genetic Manipulation in Plant Breeding, ProceedingsInternational Symposium Organized by EUCARPIA, Berlin, Germany (Sep.8-13, 1985); Thomas, W J K, et al., “Doubled haploids in breeding,” inDoubled Haploid Production in Crop Plants, Maluszynski, M., et al.(Eds.), Dordrecht, The Netherland Kluwer Academic Publishers, pp.337-349 (2003).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard (1960); Simmonds (1979); Sneep, et al. (1979); Fehr(1987)).

Uses of Sorghum

The seed of sorghum hybrid ES5200, the plant produced from seed ofES5200, a plant derived from using hybrid sorghum ES5200, and variousparts of the hybrid sorghum plant and transgenic versions of theforegoing, can be utilized for human food, livestock feed, biofuelfeedstock, biopower feedstock, and as a raw material in industry.

Sorghum is used as livestock feed, a biofuel feedstock, a biopowerfeedstock, as human food, and as raw material in industry. Sorghum grainand plant parts are used as livestock feed. Sorghum grain or productstherefrom can be consumed by humans and can be used in making analcoholic beverage. Sorghum dry milling products include, for example,grits, meal and flour. Starch and other extracts for food use can beprovided by the wet milling process. Sorghum starch and flour haveapplication in the paper and textile industries. Other industrial usesinclude applications in adhesives, building materials and as oil-wellmuds. Considerable amounts of sorghum, both grain and plant material,have been used in industrial alcohol production.

In some embodiments, plants described herein and/or hybrids therefromhave a biomass yield and/or composition that permits efficientprocessing into free sugars, and subsequently ethanol, for energyproduction. In some embodiments, such plants provide higher yields ofethanol, butanol, dimethyl ether, cellulosic gasoline, other biofuelmolecules, and/or sugar-derived co-products per kilogram of plantmaterial, relative to control plants. By providing improved yields at anequivalent or even decreased cost of production, the plants describedherein improve profitability for farmers and processors as well asdecrease costs to consumers. Biomass of sorghum described herein canalso be used in biopower applications in thermal or thermochemicalconversion processes to produce energy. In some embodiments, plantsdescribed herein and/or hybrids therefrom have a biomass yield and/orcomposition that permits efficient processing into chopped biomass,pelleted biomass, and/or bricked biomass for biopower production.

Seeds from plants described herein can be conditioned and bagged inpackaging material by means known in the art to form an article ofmanufacture. Packaging material such as paper and cloth are well knownin the art. A package of seed can have a label, e.g., a tag or labelsecured to the packaging material, a label printed on the packagingmaterial, or a label inserted within the package, that describes thenature of the seeds therein.

Tables

In Table 2, the coleoptile seedling anthocyanin coloration of sorghumhybrid ES5200 is compared to sorghum hybrid ES5201. Seeds were plantedon Sep. 11, 2009 in a greenhouse in Thousand Oaks, Calif. and data wascollected on Sep. 22, 2009. Seedlings were at the 3-leaf stage at thetime of data collection. Anthocyanin values are based on a scale from 0to 9, where 0=no anthocyanin and 9=very strong anthocyanin color. Column1 shows the plant number, column 2 shows the anthocyanin value forsorghum hybrid ES5200 and column 3 shows the anthocyanin value forsorghum hybrid ES5201. Blank cells indicate that the plant died.

Table 3 shows statistical analysis using ANOVA of the data contained inTable 2 and indicates that there were significant differences(P=0.001928) in the seedling coleoptile anthocyanin coloration betweensorghum hybrids ES5200 and ES5201.

TABLE 2 Coleoptile Seedling Anthocyanin Coloration: Anthocyanin valuesare based on a scale from 0 to 9, where 0 = no anthocyanin and 9 = verystrong anthocyanin color Plant # ES5200 ES5201 1 0 0 2 0 0 3 0 1 4 0 1 50 0 6 0 0 7 0 0 8 0 2 9 0 0 10 0 0 11 0 1 12 0 0 13 0 1 14 0 1 15 0 0 160 0 17 0 2 18 0 0 19 0 0 20 0 1 21 0 0 22 0 0 23 0 1 24 0 0 25 0 0 26 01 27 0 0 28 0 0 29 0 0 30 0 31 0 32 0 33 0

TABLE 3 ANOVA: Single Factor SUMMARY Groups Count Sum Average VarianceES5200 29 0 0 0 ES5201 33 12 0.363636 0.363636 ANOVA Source of VariationSS df MS F P-value F crit Between Groups 2.041056 1 2.041056 10.524190.001928 4.001194 Within Groups 11.63636 60 0.193939 Total 13.67742 61

In Table 4, the anthocyanin coloration of the first leaf sheath ofsorghum hybrid ES5200 is compared to sorghum hybrid ES5201. Seeds wereplanted on Sep. 11, 2009 in a greenhouse in Thousand Oaks, Calif. anddata was collected on Sep. 22, 2009. Seedlings were at the 3-leaf stageat the time of data collection. Anthocyanin values are based on a scalefrom 0 to 9, where 0=no anthocyanin and 9=very strong anthocyanin color.Column 1 shows the plant number, column 2 shows the anthocyanin valuefor sorghum hybrid ES5200 and column 3 shows the anthocyanin value forsorghum hybrid ES5201. Blank cells indicate that the plant died.

Table 5 shows the statistical analysis using ANOVA of the data containedin Table 4 and indicate that there were significant differences(P=0.012845) in the anthocyanin coloration of first leaf sheath betweensorghum hybrids ES5200 and ES5201.

TABLE 4 Anthocyanin Coloration of the First Leaf Sheath: Anthocyaninvalues are based on a scale from 0 to 9, where 0 = no anthocyanin and 9= very strong anthocyanin color Plant # ES5200 ES5201 1 1 0 2 0 1 3 1 34 0 0 5 0 2 6 0 0 7 2 1 8 1 2 9 1 0 10 0 2 11 0 2 12 1 0 13 0 1 14 0 115 1 0 16 0 0 17 1 3 18 0 0 19 0 2 20 1 2 21 0 1 22 0 2 23 0 0 24 2 0 251 2 26 0 0 27 1 1 28 0 2 29 0 0 30 2 31 1 32 0 33 1

TABLE 5 ANOVA: Single Factor SUMMARY Groups Count Sum Average VarianceES5200 29 14 0.482759 0.401478 ES5201 33 34 1.030303 0.967803 ANOVASource of Variation SS df MS F P-value F crit Between Groups 4.627633 14.627633 6.577847 0.012845 4.001194 Within Groups 42.21108 60 0.703518Total 46.83871 61

In Table 6, various characteristics of the plants of sorghum hybridES5200 are compared with sorghum hybrid ES5201. Seeds were planted inspring 2008 in Maricopa, Ariz. and data from 10 plants of each hybridfrom the center of the field was collected in September 2009. Rowspacing was about 2 feet and seed spacing was about 6 inches. Row 2shows the plant number, rows 4-7 show the number of nodes (from 6 feetfrom the base of the plant), the leaf width in centimeters, the leafsheath length in centimeters for sorghum hybrid ES5200, respectively,and rows 9-12 show the number of nodes (from 6 feet from the base of theplant), the leaf width in centimeters, the leaf sheath length incentimeters for sorghum hybrid ES5201, respectively and column 12 showsthe averages for each characteristic.

Table 7 shows the statistical analysis using a T-test (assuming unequalvariances) of the data contained in Table 6. The null hypotheses is thatthe mean differences in the number of nodes, the leaf width, the leafsheath length and the stem diameter are equal (zero difference) betweensorghum hybrids ES5200 and ES5201. Thus, the analyses indicate that forthe number of nodes, the spread in the means for the two hybrids wouldhave occurred about approximately 8.3 percent of the time (P=0.083546and within a 10% confidence level). For leaf width and leaf sheaf, thedifferences fall within a 95% confidence level (P=0.037571 and1)=0.034058, respectively). For the stem diameter, P=0.161815, which isgreater than a 10% confidence level and typically not significant.

TABLE 6 Plant Number 1 2 3 4 5 6 7 8 9 10 AVG ES5200 No. nodes 15 20 1720 19 20 23 23 23 21 20.1 (in 6 ft from base) Leaf width (cm) 9 10 8.5 89.5 9.5 9 10 9.5 8.5 9.15 Leaf sheath length (cm) 19 19.5 18 20 18 20 1917.5 19 24 19.4 Stem diameter, 1 ft from 27 32 25 28 28 31 37 30 31 2129 base (cm) ES5201 No. nodes 19 19 17 18 19 18 17 19 20 18 18.4 (in 6ft from base) Leaf width (cm) 9 7 8.5 8 9 8 9.5 8.5 9 8 8.45 Leaf sheathlength (cm) 20 23 20 22 21 21 20 23 19 21.5 21.05 Stem diameter, 1 ftfrom 30 35 25 32 27 33 32 34 37 31 31.6 base (cm)

TABLE 7 ES5200 ES5201 NODES t-Test: Two-Sample Assuming UnequalVariances Mean 20.1 18.4 Variance 6.988889 0.933333 Observations 10 10Hypothesized Mean Difference 0 df 11 t Stat 1.909965 P(T <= t) one-tail0.041273 t Critical one-tail 1.795884 P(T <= t) two-tail 0.082546 tCritical two-tail 2.200986 LEAF SHEATH LENGTH t-Test: Two-SampleAssuming Unequal Variances Mean 19.4 21.05 Variance 3.322222 1.802778Observations 10 10 Hypothesized Mean Difference 0 df 17 t Stat −2.30482P(T <= t) one-tail 0.017029 t Critical one-tail 1.739606 P(T <= t)two-tail 0.034058 t Critical two-tail 2.109819 LEAF WIDTH t-Test:Two-Sample Assuming Unequal Variances Mean 9.15 8.45 Variance 0.4472220.525 Observations 10 10 Hypothesized Mean Difference 0 df 18 t Stat2.244994 P(T <= t) one-tail 0.018785 t Critical one-tail 1.734063 P(T <=t) two-tail 0.037571 t Critical two-tail 2.100924 STEM DIAMETER t-Test:Two-Sample Assuming Unequal Variances Mean 29 31.6 Variance 18.6666712.93333 Observations 10 10 Hypothesized Mean Difference 0 df 17 t Stat−1.46261 P(T <= t) one-tail 0.080908 t Critical one-tail 1.739606 P(T <=t) two-tail 0.161815 t Critical two-tail 2.109819

DEPOSIT INFORMATION

A deposit of the Ceres, Inc. proprietary hybrid Sorghum ES5200 disclosedabove and recited in the appended claims has been made with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110. The date of deposit was Sep. 24, 2010. The deposit of 2,500seeds was taken from the same deposit maintained by Ceres, Inc. sinceprior to the filing date of this application. All restrictions will beremoved upon granting of a patent, and the deposit is intended to meetall of the requirements of 37 C.F.R. §§1.801-1.809. The ATCC AccessionNumber is PTA-11366. The deposit will be maintained in the depositoryfor a period of thirty years, or five years after the last request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A seed of hybrid sorghum designated ES5200, wherein a representative sample of seed of said hybrid was deposited under ATCC Accession No. PTA-11366.
 2. A sorghum plant, or a part thereof, produced by growing the seed of claim
 1. 3. A sorghum plant, or a part thereof, having all the physiological and morphological characteristics of hybrid ES5200, wherein a representative sample of seed of said hybrid was deposited under ATCC Accession No. PTA-11366.
 4. A tissue culture of cells produced from the plant of claim 2, wherein said cells of the tissue culture are produced from a plant part selected from the group consisting of pollen, glume, panicle, leaf, pollen, ovule, cotyledon, hypocotyl, root, root tip, pistil, anther, floret, seed, stalk, immature embryo, and rachis.
 5. A sorghum plant regenerated from the tissue culture of claim 4, wherein the regenerated plant has all the morphological and physiological characteristics of hybrid ES5200, wherein a representative sample of seed of said hybrid was deposited under ATCC Accession No. PTA-11366.
 6. A method for producing an inbred sorghum seed wherein the method comprises crossing the plant of claim 2 with a same or a different sorghum plant and self-pollinating for generations and harvesting the resultant sorghum seed.
 7. A sorghum seed produced by the method of claim
 6. 8. A method for producing a sorghum plant that contains in its genetic material one or more transgenes, wherein the method comprises crossing the sorghum plant of claim 2 with either of another sorghum plant which contains a transgene or a transformed sorghum plant of hybrid sorghum ES5200, so that the genetic material of the progeny that results from the cross contains the transgene(s) operably linked to a regulatory element.
 9. A sorghum plant produced by the method of claim
 8. 10. A method of producing an herbicide resistant sorghum plant, wherein said method comprises introducing a gene conferring herbicide resistance into the plant of claim
 2. 11. An herbicide resistant sorghum plant produced by the method of claim 10, wherein the gene confers resistance to an herbicide selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione, triazine and benzonitrile.
 12. A method of producing a pest or insect resistant sorghum plant, wherein said method comprises introducing a gene conferring pest or insect resistance into the sorghum plant of claim
 2. 13. A pest or insect resistant sorghum plant produced by the method of claim
 12. 14. The sorghum plant of claim 13, wherein the transgene encodes a Bacillus thuringiensis protein.
 15. A method of producing a disease resistant sorghum plant, wherein said method comprises introducing a gene which confers disease resistance into the sorghum plant of claim
 2. 16. A disease resistant sorghum plant produced by the method of claim
 15. 17. A method of producing a sorghum plant with modified fatty acid metabolism or modified carbohydrate metabolism, wherein the method comprises introducing a gene encoding a protein selected from the group consisting of phytase, fructosyltranferase, levansucrase, α-amylase, invertase and starch branching enzyme or encoding an antisense of stearyl-ACP desaturase into the sorghum plant of claim
 2. 18. A sorghum plant produced by the method of claim 17 having modified fatty acid metabolism or modified carbohydrate metabolism.
 19. A method for producing a seed of a plant derived from hybrid sorghum ES5200 comprising the steps of: a) crossing the plant of claim 2 with itself or a different sorghum plant; and b) allowing seed of a hybrid ES5200-derived sorghum plant to form.
 20. The method of claim 19, further comprising the steps of: c) selfing a plant grown from said hybrid ES5200-derived sorghum seed to yield additional hybrid ES5200-derived seed of step b); d) growing said additional hybrid ES5200-derived seed to produce sorghum plants; and e) repeating the growing and crossing steps of c) and d) to generate at least a first hybrid ES5200-derived sorghum plant. 